CN103940919B - A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period - Google Patents
A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period Download PDFInfo
- Publication number
- CN103940919B CN103940919B CN201310054049.0A CN201310054049A CN103940919B CN 103940919 B CN103940919 B CN 103940919B CN 201310054049 A CN201310054049 A CN 201310054049A CN 103940919 B CN103940919 B CN 103940919B
- Authority
- CN
- China
- Prior art keywords
- atractylodes rhizome
- bighead atractylodes
- disease
- lactone
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method for detecting its resistance markers in bighead atractylodes rhizome growth period.This method is disease plant and disease-resistant plant under collection nature in the bighead atractylodes rhizome soil-borne disease high-incidence season；Simultaneously, Sclerotiumrolfsii mycelia is inoculated with using root method is hindered, after adopted respectively after infection morbidity health and disease plant, using major metabolite atractylodes lactone class level in HPLC method determination sample plant, carry out group difference comparison, determine significant difference component, the screening significant metabolin relevant with bighead atractylodes rhizome resistance, and with Sclerotium rolfsii Saccardo (Sclerotium rolfsii Sacc.) for tested bacterium, carry out the virulence test of the component, prove atractylodes lactone II bacteriostasis efficacy, resistance markers are obtained with this, for bighead atractylodes rhizome evaluation of resistance, in terms of resistant variety seed selection.
Description
Technical field
The present invention relates to a kind of method for detecting resistance markers in bighead atractylodes rhizome growth period, and in particular to a kind of bighead atractylodes rhizome secondary generation
Thank to thing atractylodes lactone II as the marker detection method relevant with bighead atractylodes rhizome soil-borne disease disease resistance.
Background technology
The bighead atractylodes rhizome (Atractylodis macrocephala Koidz.) is one of large Chinese medicine of China, and whole nation priority is about
There are 20 province (urban district) introducing and plantings, about 1.3 ten thousand hectares of current cultivated area.However, in edaphic condition, cultivation condition and weather
Under condition suitable condition, up to 50% when the loss late of the soil-borne disease morbidity of the bighead atractylodes rhizome is serious, or even cause total crop failure.Bighead atractylodes rhizome soil is passed
Disease is the limitation sex factor of bighead atractylodes rhizome plant development, and it mainly has by Sclerotium rolfsii Saccardo (Sclerotium
) etc. rolfsiiSacc. pathogenic bacteria cause.For such case, bighead atractylodes rhizome anti-disease mechanism is studied, strong resistance kind, screening is found
Diseases prevention medicament, the research for resisting bighead atractylodes rhizome disease is increasingly taken seriously.
Although the country has scholar to utilize molecule labelling method to the country on the basis of the Germplasm Resource Investigation analysis of the bighead atractylodes rhizome
The germ plasm resource of different sources Rhizoma Atractylodis Macrocephalae carries out Genetic Variation Analysis, illustrates the cultivation bighead atractylodes rhizome and keeps higher genetic diversity
The basis of medicinal material fine quality and breed breeding is to maintain, the fine-variety breeding of the bighead atractylodes rhizome has good introduces a collection condition.But the bighead atractylodes rhizome with
Other medicinal plants equally lack a large amount of sequence data information so that network analysis, microarray of gene expression etc. transcribe profile
Analysis method and cDNA-ALFP technologies are dfficult to apply to the expression analysis research of its various biological function related gene, these
The heredity of biological function including the bighead atractylodes rhizome, fertility, it is disease-resistant in terms of, seriously hinder the large-scale Gene mining of the bighead atractylodes rhizome.White
It is even more so in the disease-resistant research of the medicinal plants such as art.
Plant metabolites have the amplification expressional function of disease-resistant gene, are the biochemistry phenotype of cell or tissue, generation
Thank to the provided information of thing analysis more more useful than the information that transcript profile and Proteomic analysis are provided, can more disclose gene
Relation between phenotype, finds the molecular marker of plant disease-resistant, must in the breeding for disease resistance, anti-disease mechanism research in plant
So play great effect.The present invention provides a kind of method, specify that and bighead atractylodes rhizome soil-borne disease resistance using metabolite analysis method
Relevant characteristic indication metabolin, the mark can be used in terms of bighead atractylodes rhizome evaluation of resistance, resistant variety seed selection.
The content of the invention
The invention discloses a kind of method for detecting its resistance markers in bighead atractylodes rhizome growth period.This method is passed in bighead atractylodes rhizome soil
Disease plant and disease-resistant plant under sick high-incidence season, collection nature；Meanwhile, using root method inoculation Sclerotiumrolfsii mycelia is hindered, wait to feel
Health and disease plant are adopted in hair dyeing respectively after being ill, using major metabolite atractylodes lactone class water in HPLC method determination sample plant
It is flat, group difference comparison is carried out, significant difference component is determined, the significant metabolin relevant with bighead atractylodes rhizome resistance is screened, and with
Sclerotium rolfsii Saccardo (Sclerotium rolfsii Sacc.) is tested bacterium, carries out the virulence test of the component, it was demonstrated that
Atractylodes lactone II bacteriostasis efficacy, resistance markers are obtained with this, in terms of bighead atractylodes rhizome evaluation of resistance, resistant variety seed selection.
The purpose of the present invention through the following steps that realize：
A kinds plant the bighead atractylodes rhizome, routinely carry out field management, under 5-7 month bighead atractylodes rhizome soil-borne diseases peak period collection nature
Disease-resistant plant A1 and each more than 30 plants of disease plant A2, removes soil, and low temperature drying is standby as test sample；
B adopts each 30 plants of healthy B1 and disease plant B2 respectively using root method inoculation Sclerotium rolfsii Saccardo is hindered after 1-3 weeks
More than, soil is removed, low temperature drying is standby as test sample；
C determines the content value of atractylodes lactone class in the sample of step A and step B using HPLC methods, and using single factor test T inspections
The difference of level between analysis A1 and A2, B1 and B2 groups is tested, it is determined that the obvious high expression in bighead atractylodes rhizome disease-resistant plant and healthy plant
Metabolin is atractylodes lactone II, and further uses reference substance additional method to identify this metabolin for atractylodes lactone II；
D verifies inhibitory activity of the candidate markers to bighead atractylodes rhizome pathogenic bacteria using antibacterial experiment in vitro, with the small bacterium of the white thin,tough silk of Roche
Pyrenomycetes (Sclerotium rolfsii Sacc.) is tested strain, the virulence to the pathogenic bacteria is tested respectively, with atractylodes lactone
I, atractylodes lactone III compare, and specifying atractylodes lactone II has an obvious inhibitory action, experiment confirm metabolin atractylodes lactone II be with
The relevant mark of bighead atractylodes rhizome disease resistance.
The method of above-mentioned detection mark related to resistance in the bighead atractylodes rhizome, collection bighead atractylodes rhizome disease plant and healthy plant sample
Product are preferably the bighead atractylodes rhizome soil-borne disease high-incidence season in June.
Bighead atractylodes rhizome resistance markers are obtained by above method, sought with transcription group and protein science conventional in agriculture field
The method of molecular marked compound is looked for compare, because the minor variations of gene and protein expression can be amplified on metabolin so that
Detection is easier, and expense is low, and the technology used in research is more general, and the resistance marker's metabolin filtered out is to bighead atractylodes rhizome anti-disease mechanism
And the relevant research field of breeding will have very big value.
Brief description of the drawings
The HPLC chromatogram of Fig. 1 bighead atractylodes rhizomes sample and atractylodes lactone I (A), atractylodes lactone II (B) and atractylodes lactone III (C)
(1- atractylodes lactone I, 2- atractylodes lactone II, 3- atractylodes lactone III, C- bighead atractylodes rhizome samples 220nm detections, D- bighead atractylodes rhizome samples 276nm inspections
Survey)
Fig. 2 atractylodes lactones II bacteriostasis (the 5th day inhibiting rate 77%).
Embodiment
This method is illustrated with reference to embodiment.
Embodiment
Comprising the concrete steps that in the present embodiment：
The natural sample collection experiment of A fields is carried out in Changhua Zhejiang and Tianmu Mountains of Zhejiang Province medicinal material planting base respectively.2010
Plant year mid-March the bighead atractylodes rhizome, in mid-June, 2011 southern blight onset peak period, disease-resistant plant A1 under collection natural conditions and
Each 30 plants of removals soil of disease plant A2, low temperature drying is standby as test sample.
B Field inoculations are tested
Carried out respectively in prosperousization and Tian Mu Shan Mountain medicinal material planting base.The bighead atractylodes rhizome is planted in mid-March, 2010, in 6 months 2011
Ten days, southern blight onset peak period was using root method inoculation Sclerotiumrolfsii mycelia is hindered, after adopting healthy B1 and disease plant after infection morbidity respectively
Each 30 plants of B2, removes soil, and low temperature drying is standby as test sample.
C atractylodes lactones constituents are determined
Chromatographic condition chromatographic column：Sun Fire C18 (250mm × 4.6mm, 5 μm)；Mobile phase methanol (A)-water (B), ladder
Degree elution program is 0~16min, 60%~76%A；16~18min, 76%~100%A；18~30min, 100%A.Volume
Flow 1mL/min, 25 DEG C of column temperature, atractylodes lactone I, III Detection wavelength is that 220nm, atractylodes lactone II Detection wavelengths are 276nm,
The μ L of sample size 20.Chromatogram is shown in Fig. 1.
The preparation precision of reference substance solution weighs atractylodes lactone I, II, III reference substance in right amount, is configured to methanol containing white
Art lactone I, II, III are respectively 0.266,0.100,0.341mg/mL mixed reference substance solution.
The preparation precision of need testing solution weighs bighead atractylodes rhizome sample powder 0.5g, adds after 8mL methanol, ultrasonic 30min, standing
With methanol constant volume to 10mL, shake up, obtain 50mg/mL need testing solution.
Sample determines and uses reference substance additional method to identify atractylodes lactone II.
As a result with analysis：Using the difference of level between single factor test T check analyses A1 and A2, B1 and B2 groups, as shown in Table 1,
Atractylodes lactone II expressions are significantly higher than disease plant in its disease-resistant plant of bighead atractylodes rhizome growth period, and lactone I and the bighead atractylodes rhizome in the bighead atractylodes rhizome
Lactone II I expression is between disease-resistant plant and disease plant group without significant difference.It is considered that in disease-resistant plant in the bighead atractylodes rhizome
Ester II high level is that the amplification of bighead atractylodes rhizome disease-resistant gene expression is apparent, is the key metabolites of the anti-soil-borne disease of the bighead atractylodes rhizome.Can by table 2
Know, after bighead atractylodes rhizome inoculation Sclerotiumrolfsii, significantly elevated trend is presented in healthy plant atractylodes lactone II, III expression, wherein in the bighead atractylodes rhizome
Significant difference is presented in expression of the ester II levels in healthy plant and disease plant, and atractylodes lactone I is substantially unchanged.
The content of the disease-resistant sample of the bighead atractylodes rhizome of table 1. (A1) and susceptible sample (A2) lactone
Different letters represent significant difference
After the inoculation germ of table 2. in healthy plant (B1) and disease plant (B2) sample atractylodes lactone level
Different letters represent significant difference
C in an embodiment of the present invention, is separated using the method for above-mentioned detection bighead atractylodes rhizome soil-borne disease Research of predicting markers and analyzed
Gone out the lactone mark metabolin of correlation, it was demonstrated that atractylodes lactone II expressed in disease-resistant and healthy plant apparently higher than susceptible and
Disease plant, is resistance marker's metabolin of candidate, and its In Vitro Bacteriostasis verification method step is as follows：
(a) 0.01ml various concentrations methanol mother liquor lactones I, II, III are taken respectively in 10ml culture mediums, make into 200,
100th, 50,25,12.5 μ g/ml culture medium.Toxicity test is carried out using growth rate method.Using Sclerotium rolfsii Saccardo as confession
Try strain.Aseptic condition, under each pathogen colony edge card punch beat take bacteria cake diameter be inoculated in respectively it is each processing training
Support base flat board center and connect 1 bacteria cake per ware, often handle 3 repetitions, 25 DEG C are cultivated 72 hours, and bacterium colony is measured using crossing method
Diameter, statistical analysis is carried out using DPS softwares, calculates virulence regression equation, median effective concentration and coefficient correlation.
(b) toxicity test interpretation of result, table 3 shows that atractylodes lactone II has significant suppression to Sclerotium rolfsii Saccardo
Effect is (see Fig. 2), and EC50 is 252.34 μ g/ml, and when concentration is 200 μ g/ml, inhibiting rate reaches 77.23%；Atractylodes lactone III
There is certain inhibitory action to Sclerotium rolfsii Saccardo, EC50 is 685.32 μ g/ml, the inhibiting rate when concentration is 200 μ g/ml
For 23.45%；And atractylodes lactone I on Sclerotium rolfsii Saccardo substantially without influence.
Toxicity test of the atractylodes lactone of table 3 to Sclerotiumrolfsii
Extracorporeal bacteria inhibitor test also confirms that atractylodes lactone II has significant suppression to make to the Main Pathogenic Bacteria of bighead atractylodes rhizome soil-borne disease
With, and atractylodes lactone III only shows certain inhibition in bacteriostatic test.To sum up drawing atractylodes lactone II is and the bighead atractylodes rhizome
The relevant mark of anti-soil-borne disease.
Claims (3)
1. a kind of method for detecting resistance markers in bighead atractylodes rhizome growth period, it is characterised in that：
A kinds plant the bighead atractylodes rhizome, routinely carry out field management, in disease-resistant under 5-7 month bighead atractylodes rhizome soil-borne diseases peak period collection nature
Plant A1 and each more than 30 plants of disease plant A2, removes soil, and low temperature drying is standby as test sample；
B using root method inoculation Sclerotium rolfsii Saccardo is hindered, adopted respectively after 1-3 weeks each 30 plants of healthy B1 and disease plant B2 with
On, soil is removed, low temperature drying is standby as test sample；
C determines the content value of atractylodes lactone class in the sample of step A and step B using HPLC methods, and is examined point using single factor test T
The difference of level between A1 and A2, B1 and B2 groups is analysed, it is determined that the substantially metabolism of high expression in bighead atractylodes rhizome disease-resistant plant and healthy plant
Thing is atractylodes lactone II, and further uses reference substance additional method to identify this metabolin for atractylodes lactone II；
HPLC methods determine condition be：Chromatographic condition chromatographic column：Sun Fire C18,250mm × 4.6mm, 5 μm, mobile phase first
Alcohol A- water B, gradient elution program is 0~16min, 60%~76%A；16~18min, 76%~100%A；18~30min,
100%A；Volume flow 1mL/min, 25 DEG C of column temperature, atractylodes lactone I, III Detection wavelength is that 220nm, atractylodes lactone II detect ripple
A length of 276nm, the μ L of sample size 20；The preparation of reference substance solution：Precision weighs atractylodes lactone I, II, III reference substance in right amount, uses first
It is respectively 0.266 that alcohol, which is configured to containing atractylodes lactone I, II, III, 0.100,0.341mg/mL mixed reference substance solution；For examination
The preparation of product solution：Precision weighs bighead atractylodes rhizome sample powder 0.5g, and methanol constant volume is used after adding 8mL methanol, ultrasonic 30min, standing
To 10mL, shake up, obtain 50mg/mL need testing solution；
D verifies inhibitory activity of the candidate markers to bighead atractylodes rhizome pathogenic bacteria using antibacterial experiment in vitro, with Sclerotium rolfsii Saccardo
For tested strain, the virulence to the pathogenic bacteria is tested respectively, is compared with atractylodes lactone I, atractylodes lactone III, specifies atractylodes lactone
II has obvious inhibitory action, and experiment confirms that metabolin atractylodes lactone II is the mark relevant with bighead atractylodes rhizome disease resistance；
Wherein In Vitro Bacteriostasis verification method step is as follows：Take respectively 0.01ml various concentrations methanol mother liquor lactone I, II, III in
In 10ml culture mediums, make into 200,100,50,25,12.5 μ g/ml culture medium；Toxicity test is carried out using growth rate method；
Using Sclerotium rolfsii Saccardo as trying strain；Being beaten under aseptic condition in each pathogen colony edge card punch takes bacteria cake straight
Footpath is inoculated in each processing culture medium flat plate center and connects 1 bacteria cake per ware respectively, often handles 3 repetitions, and 25 DEG C are cultivated 72 hours, are adopted
Colony diameter is measured with crossing method, statistical analysis is carried out using DPS softwares, virulence regression equation, median effective concentration is calculated
And coefficient correlation.
2. the method according to claim 1 for detecting resistance markers in bighead atractylodes rhizome growth period, it is characterised in that：Atractylodes lactone
II substantially high expression, and related to the anti-soil-borne disease of the bighead atractylodes rhizome in the bighead atractylodes rhizome with resistance against diseases.
3. the method according to claim 1 for detecting resistance markers in bighead atractylodes rhizome growth period, it is characterised in that：Gather the bighead atractylodes rhizome
Disease plant and healthy plant sample are the bighead atractylodes rhizome soil-borne disease high-incidence season in June.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310054049.0A CN103940919B (en) | 2013-01-19 | 2013-01-19 | A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310054049.0A CN103940919B (en) | 2013-01-19 | 2013-01-19 | A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103940919A CN103940919A (en) | 2014-07-23 |
| CN103940919B true CN103940919B (en) | 2017-08-25 |
Family
ID=51188673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310054049.0A Expired - Fee Related CN103940919B (en) | 2013-01-19 | 2013-01-19 | A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103940919B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796884A (en) * | 2017-09-06 | 2018-03-13 | 中国农业大学 | A kind of bactericide study on mechanism method based on metabolism group |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507356B (en) * | 2018-12-30 | 2020-01-07 | 鲁南制药集团股份有限公司 | Quality detection method of 'Hui Tongbiang' capsule |
| CN115097053B (en) * | 2022-06-21 | 2023-05-02 | 山东农业大学 | Metabolic marker for identifying phytophthora sojae root rot disease inductive reactance condition and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6635490B1 (en) * | 2000-05-25 | 2003-10-21 | Noble Laboratories | Procedure for the simultaneous quantitative and qualitative analysis of both flavonoid glycosides and steroidal glycosides |
| CN102680631B (en) * | 2012-05-24 | 2014-02-26 | 成都中医药大学 | Detection method for atractylodes macrocephala koidz medicinal materials |
-
2013
- 2013-01-19 CN CN201310054049.0A patent/CN103940919B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796884A (en) * | 2017-09-06 | 2018-03-13 | 中国农业大学 | A kind of bactericide study on mechanism method based on metabolism group |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103940919A (en) | 2014-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Minocha et al. | Development of a standardized methodology for quantifying total chlorophyll and carotenoids from foliage of hardwood and conifer tree species | |
| Paez-Garcia et al. | Root traits and phenotyping strategies for plant improvement | |
| Wu et al. | Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markers | |
| Weston et al. | Metabolic profiling: an overview-new approaches for the detection and functional analysis of biologically active secondary plant products | |
| Sharma et al. | Rapid in situ analysis of plant emission for disease diagnosis using a portable gas chromatography device | |
| Gomez et al. | Molecular and phenotypic diversity of common bean landraces from Nicaragua | |
| Kjellsson et al. | Methods for risk assessment of transgenic plants: I. Competition, establishment and ecosystem effects | |
| Laothawornkitkul et al. | Volatile organic compounds as a diagnostic marker of late blight infected potato plants: A pilot study | |
| Miedaner et al. | Diversity, spatial variation, and temporal dynamics of virulences in the German leaf rust (Puccinia recondita f. sp. secalis) population in winter rye | |
| CN103940919B (en) | A kind of method for detecting resistance markers in bighead atractylodes rhizome growth period | |
| CN105274238A (en) | Screening method of chilo suppressalis reference genes under stress of chlorantraniliprole and application of chilo suppressalis reference genes | |
| Zhu et al. | High γ-aminobutyric acid content, a novel component associated with resistance to abamectin in Tetranychus cinnabarinus (Boisduval) | |
| Reveglia et al. | Production of phytotoxic metabolites by Botryosphaeriaceae in naturally infected and artificially inoculated grapevines | |
| York et al. | Bioenergy Underground: Challenges and opportunities for phenotyping roots and the microbiome for sustainable bioenergy crop production | |
| Feng et al. | Quantitative trait loci analysis of phenolic acids contents in Salvia miltiorrhiza based on genomic simple sequence repeat markers | |
| Naim-Feil et al. | The Cannabis Plant as a Complex System: interrelationships between cannabinoid compositions, morphological, physiological and phenological traits | |
| Kudo et al. | Seasonal variation of alkaloids and polyphenol in Ephedra sinica cultivated in Japan and controlling factors | |
| JP5920827B2 (en) | Identification method of disease resistant variety line | |
| Mo et al. | Identification of phenological growth stages of four Morus species based on the extended BBCH-Scale and its application in fruit development with morphological profiles and color characteristics | |
| CN105713959B (en) | Molecular detecting method of the diamondback moth to avermectin target resistance | |
| Menegat et al. | Chlorophyll fluorescence microscreening as a rapid detection method for herbicide resistance in grass weeds in North China Plain winter wheat production systems and beyond. | |
| Zhu et al. | Relationship of carbon isotope discrimination with biomass and water use efficiency for alfalfa in northwestern China | |
| CN101985652A (en) | High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency | |
| Song et al. | Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl. | |
| Yang et al. | Rhizosphere microbial markers (micro-markers): A new physical examination indicator for traditional Chinese medicines |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170825 Termination date: 20190119 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |